Naphthalene derived chromogenic enzyme substrates

ABSTRACT

Conjugates of 2,3-dihydroxynaphthalene and its derivatives with enzyme cleavable groups are chromogenic substrates that form colored compounds when complexed with metal ions, e.g. iron ions, on cleavage by enzymes, and are useful in microbial detection and identification. The cleavage products form purple or red-brown colored complexes, that can easily be observed by the naked eye. Microbes can be grown in the presence of the substrates and the metal salts that provide the metal ion for complexing with the 2,3-dihydroxynaphthalene product. Substituents in the naphthalene ring may affect the solubility of the substrates and also the diffusibility and color of the metal complexes. Some of the substrates yield soluble complexes on cleavage and are of particular value in liquid growth media. Other substrates produce less soluble complexes that are more suitable for use in solid agar media. 
     Some substrates are new compounds, such as those having the general formula II 
     
       
         
         
             
             
         
       
         
         
           
             wherein one of the following applies 
             i) m=0, R 4 ═R 5 =Z 1 ═H, Y 1  is selected from the group consisting of D-glucuronyl and D-ribofuranosyl; 
             ii) m=2, each R 6  is Br, R 4 ═R 5 ═H or Br, Z 1 ═H, Y 1  is glycosyl or phosphate; 
             iii) m=1, R 6  is —SO 3 X, X is H or M +  wherein M +  is an alkali metal cation or a non-metal cation, Y 1  is glycosyl and R 4 ═R 5 =Z 1 ═H; 
             iv) m=0, R 4 ═NO 2 , R 5 ═Z 1 ═H, Y 1 =glycosyl. 
           
         
       
    
     Methods of synthesizing the substrates are described.

FIELD OF USE

This invention concerns the application of artificial chromogenic enzymesubstrates for the detection and identification of microorganisms.

BACKGROUND TO THE INVENTION

Most artificial substrates for hydrolytic enzymes in current large-scaleapplications in diagnostic microbiology are either chromogenic orfluorogenic. Fluorogenic assays suffer from certain disadvantages,including intrinsic background fluorescence from certain samples.However, perhaps their main disadvantage is the need to use a lamp orother source of UV light to generate the fluorescence. Chromogenicenzyme substrates have an advantage in that the endpoint can bedetermined with the naked eye. Alternatively, the released chromogen maybe assayed using simple spectrophotometers working by absorption oflight in the visible wavelengths. For an enzyme substrate to be of realvalue in diagnostic microbiology, certain conditions need to be met.When attached to the target residue (i.e. a sugar, ester or phosphate)the artificial enzyme substrates should be practically colourless orhave a very low background colouration that does not interferesignificantly with the test procedure. However, once cleaved from thetarget portion by enzymatic hydrolysis, the free core molecule is eitherhighly coloured or can be converted to a coloured compound in situ byfurther chemical (i.e., non-enzymatic) reaction. Ideally, this reactionshould be virtually instantaneous with the enzymatic cleavage and theconditions or reagents required to produce the colour should preferablybe already present in the media, and therefore must be able to allowadequate microbial growth. Under these conditions, the presence of thecoloured end-product gives a good indication of the enzyme activitytargeted. The substrates should be convenient to synthesise frominexpensive starting materials so that many different substrates can beproduced from the same core molecule; they should be easy to use,suitable for continuous assays, and they should be able to work underboth aerobic and anaerobic conditions. Although not a prerequisite forultimate utility, it would be a further advantage if the chromogen wascontrasting in colour to the chromogens of currently available enzymesubstrates. In liquid media the chromogen should be largely soluble. Insolid or gelled plate media (such as the commonly used agar plates) thechromogen should be non-diffusible so that the colour remainsconcentrated in the colony mass. In agar tube media, diffusion of thechromogen is acceptable.

Many different artificial chromogenic enzyme substrates derived fromvarious core molecules have been produced and are currently commerciallyavailable. However, all core molecules have limitations as well asadvantages depending upon the specific application. Some of the positiveand negative attributes of the main types of chromogenic enzymesubstrates employed to detect glycosidase activities may be set out asfollows.

Nitrophenyl substrates are widely employed in liquid media. One commonexample, o-nitrophenyl-β-D-galactopyranoside (ONPG), is cheap and easyto use. However, in agar plate media diffusion of the yellowo-nitrophenol chromogen makes it impractical to detect enzyme-positivefrom enzyme-negative cultures of microorganisms in a polymicrobialculture. A further disadvantage is that the pale yellow colour givenafter hydrolysis is not dissimilar to the background colour alreadypresent in certain culture media. Moreover, the maximum colour ofo-nitrophenol is only generated at highly alkaline pH at which mostmicroorganisms will not grow. p-Nitrophenyl-β-D-glucuronide is an enzymesubstrate that has been used to detect β-D-glucuronidase from E coli andthereby identify this bacterium, but the yellow p-nitrophenol shares allthe defects of its isomer o-nitrophenol. Phenolphthalein is anotherinexpensive core molecule of some chromogenic enzyme substrates. As withthe nitrophenols, the phenolphthalein aglycone diffuses greatly in agarmedia. Moreover, the free phenolphthalein has to be made highly basicbefore the red colour develops, so phenolphthalein substrates areunsuitable for continuous assays. Although this core molecule isinexpensive, its glycosides, such as phenolphthalein-β-D-glucuronide,are very expensive, undoubtedly because of difficulties with theirsynthesis. For all the above reasons, phenolphthalein-derived enzymesubstrates are little used currently. Resorufin is a very costly coremolecule that is both chromogenic and fluorogenic; the few commerciallyavailable glycosides derived from it are thus very expensive. Resorufinsubstrates are suitable for liquid media only.

For use in solid media (agar plates) and other situations in which anessentially insoluble or non-diffusible chromogenic endpoint isrequired, indoxyl substrates tend to be preferred. X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) is a common example.On enzymatic cleavage, oxidation of 5-bromo-4-chloroindoxyl in situyields a bright blue-green insoluble indigo dye that will stay localisedwithin microbial colonies, thereby affording good differentiation ofcolonies within a polymicrobial culture on agar plates.X-β-D-glucuronide is extensively used in microbiological media to detectE. coli. A whole range of differently substituted indoxyl glycosides,carboxylic acid esters and phosphates is commercially available. The useof different coloured indoxyl substrates in the same medium has beenexploited for the simultaneous detection of two or more different enzymeactivities [J. N. Roth and W. J. Ferguson, U.S. Pat. No. 5,219,922,(1993); W. J. Ferguson, U.S. Pat. No. 5,358,854, (1994)]. Negativeaspects of indoxyl substrates are that they are not well suited toliquid media and that the indigo dye can only be generated underoxidative conditions. This last property makes them unsuited to thedetection of anaerobes and is a severe limitation when dealing with thistype of organism. Indoxyl substrates have also been shown to exhibittoxicity to bacteria as evidenced by the formation of small colonies [A.S. Baron and F. E. Nano, Mol. Microbiol., 29, 247-259, (1998); L.Butterworth et al, J. Appl. Microbiol., 96, 170-176, (2004)]. Typically,indoxyl substrates are more expensive than their nitrophenylcounterparts; X-Gal is approximately one order of magnitude moreexpensive than ONPG in bulk.

Enzyme substrates derived from 1-naphthol, 2-naphthol, and compounds inthe naphthol-AS series are known. The released naphthols are notthemselves chromogenic, and a diazonium salt has to be added to producethe insoluble dye. The need to add a coupling reagent post-incubationrenders these substrates unsuitable for continuous assay as well asgreatly adding to the inconvenience of any test. Above all, diazoniumsalts are to be avoided for routine work because of their generaltoxicity.

James and co-workers [A. L. James et al, Applied and EnvironmentalMicrobiology, 66, 5521-5523, (2000)] synthesised the novel chromogenicsubstrate p-naphtholbenzein-β-D-galactopyranoside for use in solid platemedia. Bacterial hydrolysis of this substrate gave pink, non-diffusiblecolonies. The principle on which this aglycone works appears to be itslarge size combined with its hydrophobic nature. Its sensitivity whenchallenged with nearly 400 bacterial strains was lower than X-Gal. Thismay explain why the substrate is not commercially available and whyother glycosides of p-naphtholbenzein have not so far been reported.

Recently, substrates generating their colour via intermolecular orintramolecular aldol reactions have been disclosed [U. Spitz et al, EP20090159639, (2009)]. Although a number of advantages are claimed forthese Aldol substrates, their synthesis actually starts from otherindoxyls substrates, thus making them potentially very expensive.Another disadvantage of their synthesis is that some examples requirethe preparation and use of some reagents not commercially available.

Artificial chromogenic enzyme substrates based on metal chelation arewell-known. Glycosides of 8-hydroxyquinoline generate insoluble colourediron chelates after release of the aglycone. A commercial medium using8-hydroxyquinoline-β-D-glucuronide has been evaluated [R. D. Reinders etal, Lett. Appl. Microbiol., 30, 411-414, (2000)], but for bacterialdiagnosis a restricting factor is the toxicity of the aglycone toGram-positive organisms [J. D. Perry et al, J. Appl. Microbiol., 102,410-415, (2007)]. The range of 8-hydroxyquinoline substratescommercially available is limited, possibly on account of difficultiesin their synthesis, and the substrates are unsuited to liquid media.However, most of the artificial enzyme substrates described as workingvia metal chelation are composed of core molecules that contain anortho-dihydroxyaromatic system. Many different compounds containing theortho-dihydroxyaromatic system have been described as metal chelators,and they form coloured complexes with a wide variety of metal ionsdepending on the compound and the metal ion in question. Nevertheless,the first chromogenic chelating-type enzyme substrate containing anortho-dihydroxyaromatic moiety to be used was a natural compound,esculin. Esculin is the β-D-glucopyranoside of esculetin. Used with aniron salt, esculin finds employment as a reagent for Group Dstreptococci [A. Swan, J. Clin. Path., 7, 160-163, (1954)].Unfortunately, extensive diffusion of the esculetin-iron chromogenpresents a problem on agar plate media. The core molecule esculetin isexpensive, and this has undoubtedly blocked the commercial developmentof further glycosides made from it. Therefore esculetin glycosides arenot ideal substrates.

In order to address some of the aforementioned limitations of both theindoxyl enzyme substrates and the existing substrates working by metalchelation, James and Armstrong devised novel chromogenic enzymesubstrates, the lead core molecule being cyclohexenoesculetin (CHE) [A.L. James and L. Armstrong, U.S. Pat. No. 6,008,008, (1999)]. CHEcontains the ortho-dihydroxyaromatic moiety. CHE substrates present nodiffusion, and employing them to detect bacteria on solid plate mediaproduces discrete black colonies in the presence of iron, which greatlyassists in identification in mixed cultures. CHE enzyme substrates haveno background coloration, do not show any measurable toxicity tomicroorganisms and they can be used under both anaerobic and aerobicconditions. The intense black colour of the CHE-iron chelate may even beused to good effect to mask the colour generated by indoxyl substrates,depending on the circumstances [J. D. Perry et al, J. Clin. Microbiol.,37, 766-768, (1999)]. One disadvantage of CHE glycosides is the relativeexpense of synthesising CHE itself. Metal chelating-type enzymesubstrates with the ortho-dihydroxyaromatic system have also been madefrom the well-known dye alizarin [L. Armstrong and A. James, U.S. Pat.No. 7,052,863 (2006) and U.S. Pat. No. 7,563,592 (2009)].Alizarin-β-D-galactopyranoside was shown to be a highly sensitivesubstrate in agar plate media with an optimal concentration just overhalf that of X-Gal in the application studied, and this concentrationwas very much less than that required for the galactosides of CHE and8-hydroxyquinoline [A. L. James et al, Letters Appl. Microbiol., 30,336, (2000)]. Another useful attribute of this substrate is its abilityto form different colours of chelate depending on the metal ion used. Inagar media, alizarin substrates give a violet colour with iron and abright pink chelate with aluminium. However, the toxicity of alizaringives rise to small colonies [J. D. Perry et al, J. Appl. Microbiol.,102, 410 (2006)].

Yet a further class of enzyme substrates employing metal chelation withan ortho-dihydroxyaromatic system has been disclosed [M. Burton, EP1438423, (2007)]. The essence of these substrates is that theortho-dihydroxybenzene ring is not fused to any other ring system,therefore they are catechols. Other groups (if any) are attached to thecatechol ring by single bonds. Substrates of the parent compound,catechol itself, generate a fairly intense black chelate in the presenceof iron salts after enzymatic hydrolysis, although this chelate is proneto diffuse in agar media. Catechol β-D-ribofuranoside was demonstratedas an effective enzyme substrate for the revelation ofβ-D-ribofuranosidase activity in Shigella and Salmonella, but diffusionof the chromogen would appear to limit its use on agar plate media. Thecatechol-derivative 3′,4′-dihydroxyflavone (DHF) affords substratesshowing little or no diffusion in agar media. Like CHE, DHF substratesyield dark brown or black iron chelates with iron salts, but they showsome advantage over CHE substrates in that they can form a yellow,non-diffusible chelate with aluminium salts. DHF substrates areessentially non-toxic to microorganisms, but the DHF aglycone isdifficult to synthesise and, although it is commercially available, itis very expensive.

WO2008/004788 discloses the synthesis of the dicaprylate of2,3-dihydroxynaphthalene (DHN). The compounds are said to have potentialtherapeutic utility for treating the skin disease caused by excessiveproduction of melanin.

Bogdanov et al in Phosphorous, Sulphur and Silicon (2008) 183:650-651describe synthesis of monophosphate esters of DHN and a ring brominatedanalogue. Uses of the esters are not disclosed.

GB2022267 discloses DHN conjugates for use in photographyin conjunctionwith chromogenic compounds. The DHN conjugates may decompose under theaction of thermal energy, or by reaction with gaseous or liquidchemicals, to then react with the chromogenic compounds to form acoloured image.

At the present time, there is no class of chromogenic enzyme substratesthat is inexpensive and simple to prepare, is easy to use, and issuitable for continuous assays of microorganisms in both liquid andsolid media, and under both aerobic and anaerobic conditions.

SUMMARY OF THE INVENTION

This invention relates to the use of chromogenic enzyme substrates wherethe core molecule is provided by 2,3-dihydroxynaphthalene (DHN) and someof its simple derivatives.

According to the first aspect of the present invention, there isprovided a new method of detecting target enzyme activity in a mediumcomprising the steps:

a) contacting a metal compound and an enzyme substrate of the generalformula I with a substance suspected of containing or producing saidtarget enzyme

wherein Y is an enzyme cleavable group;

Z is H, or a metal cation or non-metal cation, acyl or the same enzymecleavable group as Y;

R² and R³ are each independent selected from the group consisting of H,OH, C₁-C₈ alkyl, C₂-C₂₄ acyl, halogen and nitro, provided that if Z is Hor a salt, then R² must not be OH;

R¹ is C₁-C₈ alkyl, halogen, NO₂, C₂-C₂₄ acyl, OH or —SO₃X, where X is H,a metal ion or a non-metal cation;

n is 0-4;

such that a product of enzymatic substrate cleavage is capable ofchelating the metal ion of the said metal compound, thereby forming acoloured compound; and

b) detecting the presence of the coloured compound.

According to a second aspect of the present invention a new compositionfor microbial growth contains the substrate of formula I, the metalcompound as defined above and microbial growth nutrients. Thecompositions may be growth media, or may be added to other components,or alternatively diluted with water or buffer, to form the microbialgrowth medium.

According to a third aspect of the invention, the composition containinga mixture of the substrate of formula I and an iron compound is believedto be new. This preferred composition may be added to other chromogenicmicrobial growth medium to form a suitable medium for growing microbesto detect the presence of the selected enzyme activity. Alternatively,the mixture may have other utilities, for instance in chromogenicanalysis of enzymes not requiring microbial growth in the presence ofthe substrate, or to contact samples believed to contain said enzymesfrom a microbial or non-microbial source.

In the above aspects of the invention, the right hand ring of the2,3-dihydroxynaphthalene (DHN) core may be unsubstituted, i.e. n is 0.In other embodiments n is 1 and the substituent R¹ confers useful(increased or decreased) solubility or diffusibility characteristics. Toprovide increased water solubility, particularly useful for substratesfor lipase and esterase which may otherwise be relatively hydrophobic,R¹ may be SO₃X where X is H or M^(p+) _(1/p), wherein p is 1, 2 or 3,and M^(p+) is a metal ion. Preferably, M is Na, K or Rb (and p is 1).Alternatively M^(p+) may be an alkaline earth metal, i.e. p is 2. Toprovide reduced solubility, or diffusibility in solid media the ring maybe substituted by bromine atoms, and/or R² and R³ may be bromine.

The cleavable group Y is selected according to the target enzyme. Forglycosidase targets the group is a glycosyl group, such as a glucosyl,ribofuranosyl, galactosyl or glucuronyl. For phosphatase, the group isphosphate. For lipase or esterase the group is an acyl group such as aC₂₋₂₄ acyl, for instance capryl. A glucuronyl group may be in the formof the free acid or an ester, or in the salt form with a metal cation ornon-metal cation. Non-metal cations encompass inorganic cations, such asNH⁺ ₄, and organic cations.

The group Z may also be enzyme cleavable, in which case it is usuallythe same as Y. Acyl ester substrates for lipases and esterases may bedi-substituted, for instance. Where Z is acyl and is a different groupto Y, it will need to be cleaved in order for the DHN product to chelatethe metal ion and form the coloured compound. Such a cleavage reactionmay be enzymatic or non-enzymatic.

The metal compound is selected to have a metal ion which forms acoloured compound (believed to be a coordination complex) with thecleavage product (and which is not coloured or is distinguishablycoloured from that complex). The metal compound is preferably non-toxicfor microorganisms, so that, where the test method involves a step ofincubating a sample to allow growth of microorganisms, this step may becarried out in the presence of the metal compound. Alternatively themetal compound may be added later.

The composition of the invention may itself be a growth medium or may bea premix for adding to a growth medium, or a concentrate from which agrowth medium, usually a liquid growth medium, may be formed, e.g. bydilution.

The medium generally contains nutrients for microbial growth, growthpromoters, growth inhibitors and/or other substrates, preferablychromogenic substrates, for other microbial enzymes. We have found thatthe enzyme substrates defined herein and their cleavage products allowmicrobes to grow so that microbial growth can be carried out in thepresence of those compounds. The metal compound is also selected for itscompatibility with microbial growth. It is thus not necessary for eitherthe substrate or metal compound to be added after a sample has beenincubated such that putative enzyme activity is generated upon microbialgrowth. Rather the incubation medium can contain both compounds at thestart of incubation.

The growth medium in some aspects of the present invention is a liquidgrowth medium, i.e. any medium that is suitable for microbial growth. Inthis specification reference to liquid means liquid according to theconventional sense of the word, and would be understood by the skilledperson to mean free flowing or capable of being poured. In this contextliquid media can also refer to viscous liquids, viscosified to provideeasier handling and resistance to spillage from the incubation vessel insuch an assay. Such viscosity can result from, for example, the additionof agar or other gelling agents in amounts too low to form conventionalplate media. Concentrations of agar less than 0.5% should be effectivein the liquid media used in the present invention.

The above method is distinguished from plate assays in that it isunnecessary to pinpoint individual colonies, and therefore the medium isnot required to be so solid as to maintain colonies in a singleorientation.

The present invention may also be used for solid microbial growth media,such as agar gel plate media, whereon pinpoint colonies are maintainedand may be incubated upside down because the concentration of agar issufficient to create and maintain a firm gel.

The microbes for which the present invention is particularly suited,generally comprise bacteria to be detected. Microbial and bacterialgrowth media are composed of various nutrients to support the growth ofthe microbial cultures. Such nutrients may include a carbon source,nitrogen source, a source of usable potassium, amino acids, salts,vitamins and their cofactors, metabolic intermediates and minerals.

Carbon sources may include tryptone, peptone, casein and sugars,preferably lactose and glucose.

Nitrogen sources may include amino acids, tryptone, peptone, caseinextract, and ammonium sulphate.

Salts may include ferric chloride, copper sulfate, manganese sulfate,potassium chloride, potassium iodide, zinc sulfate, magnesium chloride,potassium phosphate monobasic, potassium phosphate dibasic, sodiumcarbonate, magnesium sulfate, sodium chloride, calcium chloride andsodium pyruvate.

Vitamins may include biotin, pantothenate, folic acid, inositol,p-aminobenzoic acid, pyridoxine hydrochloride, riboflavin and thiamine.

A common source of amino acids, vitamins and minerals, as well as carbonand nitrogen, is yeast extract, which may form part of the growthmedium. Blood may also be used to supplement growth media with necessarynutrients.

Additionally, a microbial growth medium may contain antibacterial orantifungal compounds to aid in selecting and amplifying the microbes ofinterest.

Detergents may be included to act as dispersing agents, without anyantibacterial activity.

To generate a colour, the cleaved diol must chelate an ion, which isderived from a metal compound. The metal compound is preferably an ironcompound and most preferably a water soluble iron salt of an organic orinorganic acid, e.g. iron (III) ammonium citrate, iron(II) gluconate,iron(II) acetate, iron(II) citrate, or iron(II) chloride. The ironcompound can be iron(II) or iron(III). It will be appreciated by theskilled person that at least trace quantities of iron may be present inmedia or samples; however, it is usually necessary to supplement themedium with sufficient quantities of an iron compound for the inventionto work. The concentration of iron in the growth medium is preferably0.2 to 1 g/l.

It may be desirable to use the method in conjunction with a substratecapable of detecting another enzyme activity, preferably a nitrophenylderivative. It is therefore important that the colour resulting from theother test does not interfere with or mask the colour produced by theDHN substrate. It was found that the pale yellow colour of the ONPGsubstrate does not interfere with visualising the darker colour of theDHN substrate upon cleavage.

The method of the first aspect of the invention is useful for detectingthe presence of microbes, for instance microbial contamination of food,drinks, or water that will come into contact with humans or animals.Particularly useful substrates in the method are for detectingglucuronidase or galactosidase which are useful microbial markers. Themethod may be carried out according to routine test protocols and usingstandard sampling methods, and growth stages. The results are observedvisually or by machine, usually with incident visible light. Incubationmay be overnight, but for samples with high levels of enzyme alreadypresent shorter incubation may be sufficient, for instance after 1hour's incubation, sometimes 4-6 hours incubation. Incubationtemperatures are selected according to the species being detected andmay be at room temperature, but is more often at raised temperature inthe range of 30 to 50° C., preferably in the range 35 to 45° C.

Some of the enzyme substrates are new. According to a fourth aspect ofthe invention there is provided an enzyme substrate of the generalformula II

-   -   wherein one of the following applies    -   i) m=0, R⁴═R⁵=Z¹═H, Y¹ is selected from the group consisting of        D-glucuronyl and D-ribofuranosyl;    -   ii) m=2, each R⁶ is Br, R⁴═R⁵═H or Br, Z¹═H, Y¹ is glycosyl or        phosphate;    -   iii) m=1, R⁶ is —SO₃X, X is H or M⁺ wherein M⁺ is alkali metal        ion or a non-metal ion, Y¹ is glycosyl and R⁴═R⁵=Z¹═H;    -   iv) m=0, R⁴═NO₂, R⁵═Z¹═H, Y¹=glycosyl.

These novel substrates are utilised in compositions with a metalcomponent. The novel compounds are made from DHN or protected orderivatised versions thereof, and hydroxyl-group containing startingmaterials or acids, using conventional conjugation chemistry withnecessary protection/deprotection of functional groups.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Among the compounds possessing the ortho-dihydroxyaromatic system, DHNand DHN-6-sulfate are well-known metal chelators. They form colouredcomplexes with a variety of different metals, including first rowtransition metals such as iron, titanium and vanadium [V. Patrovsky,Coll. Czech. Chem. Commun., 35, 1599-1604, (1970)]. The use oftransition metal complexes of DHN, DHN-6-sulfate and some relatedortho-dihydroxyaromatic compounds as potential or actual analyticalreagents has been reviewed [P. K. Tarafder and R. K. Mondal, Rev. Anal.Chem., 30, 73-81, (2011)]. Until the present invention, DHN and itssimple derivatives have not been considered as the core molecule part ofchromogenic enzyme substrates. When the DHN substrates of the presentinvention are incorporated into microbiological growth media containingan iron compound, enzymatic cleavage by microorganisms liberates thefree DHN molecule. This reacts spontaneously with the iron compound togenerate a highly coloured chelate that allows easy detection of anyenzyme-positive reactions by eye (i.e., by the means of incident visiblelight). The colour given by the reaction depends upon the substitutionpattern of the DHN-derivative, and may also be influenced by pH and themix of oxidation states of the iron, as well as the presence of othercomponents contained in the growth medium. The colour may be describedvariously as brown, purple or maroon. However, whatever its hue, thecolour is completely different to that of most microbiological growthmedia and is intense enough to allow unambiguous detection ofenzyme-positive reactions.

Until the present work, only a very limited number of DHN derivativeshaving the potential to act as chromogenic enzyme substrates had beenmade, although DHN itself has been known for over 100 years. It is onlymuch more recently that glycosides of DHN have been synthesised.Ellervik and co-workers described the synthesis ofDHN-β-D-xylopyranoside [M. Jacobsson and U. Ellervik, TetrahedronLetters, 43, 6549-6552, (2002)] and DHN-di-β-D-xylopyranoside [R.Johnsson et al, Bioorganic and Medicinal Chemistry, 15, 2868-2877,(2007)] as part of a large set of other compounds to investigate theirantiproliferative effects on cancer cells. Sakuma and Yokoe describedthe preparation of DHN-β-D-glucopyranoside (4) [K. Sakuma and I. Yokoe,Japanese Patent Publication No. 2004-224763, (2004)]. This compound wasadvocated as a potential bleaching agent for use in cosmetics [K. Sakumaand I. Yokoe, Japanese Patent Publication No. 2004-224762, (2004)].Clearly, none of the aforementioned prior art concerning DHN compoundsis pertinent to the present application wherein they are employed aschromogenic enzyme substrates for the detection of microorganisms. Noris the more recent technique of Bhowmik and Maitra [(S. Bhowmik and U.Maitra, Chem. Commun., 48, 4624-4626, (2012)] wherein these authorsdescribed the development of a time-delayed luminescence method fordetecting enzyme activity using terbium(III) acetate in conjunction witheither DHN-β-D-glucopyranoside (4) or DHN-diesters. This method differsgreatly from the present invention in several important respects.Firstly, it is a luminescence method in which the luminescence istriggered by excitation of the sample with ultraviolet light, whereasthe present invention is a chromogenic method involving absorbance oflight in the visible spectrum. Bhowmik and Maitra reported that theirtechnique (which they state does not involve chelation) worksexclusively in a gel medium, and this is a further major difference fromthe present invention which works very well in a fluid or liquid mediumas well as in agar gels. A third important difference is that thenecessary gelling agent of Bhowmik and Maitra was the sodium salt of abile acid, cholic acid. Cholic acid and other bile acids have long beenknown to inhibit the growth of certain microorganisms [Binder et al,Amer. J. Clin. Nutr., 28, 119-125, (1975); Kurdi et al, J. Bacteriol.,188, 1979-1986, (2006)]. The concentration of sodium cholate used byBhowmik and Maitra was 15 mM, and there was no suggestion by theseauthors that microorganisms are able to grow in or on their reactionmedium. In this context it should be noted that the growth of someintestinal bacteria is completely suppressed with cholic acidconcentrations lower than 15 mM [Floch et al, Amer. J. Clin. Nutr., 25,1418-1426, (1972)]. Additionally, it should be remarked that the presentinvention requires the incorporation of metal compounds, e.g. firsttransition metal series metal compounds, preferentially iron compounds,into the medium. The iron compounds may be either iron(II) or iron(III);or a mixture of both states. In some instances, if other metal ions aresubstituted for iron, including ions such as Ti(IV) that are known toform coloured complexes with DHN, then little or no visible colour isdeveloped after enzymatic hydrolysis. Similarly, using the lanthanideterbium(III) acetate instead of an iron compound does not furnish acoloured end-product. The metal compound used in the invention musttherefore be selected having regard to the colour-forming capabilitywith the cleaved product of the enzymatic reaction.

Substitution products of the DHN core molecule yield both enzymesubstrates and endpoints of very different solubilities, and this is afurther useful feature of the present invention. Thus, unsubstituted-DHNcompounds are fairly water-soluble and the soluble DHN-iron chelate isbest suited to liquid growth media. Compounds derived from DHN-6-sulfateare even more water soluble and may be preferred when the solubility ofthe enzyme substrates in aqueous media is problematic, for instance inthe development of assays for esterases such as lipase which requiresubstrates that are derivatives of fatty acids and may have limitedwater solubility unless derivatised by ionic groups elsewhere in themolecule. In contrast, substrates based on 6,7-dibromo-DHN (as describedbelow) give insoluble precipitates with iron compounds and they areparticularly suited to agar plate media, where they are able tovisualise single colonies.

Preferred and non-limiting aspects of the invention are described inmore detail below, which includes worked examples of microbial testmethods using chromogenic substrates, followed by synthetic methods usedto make the substrates.

Example 1—Testing of DHN-β-D-Galactopyranoside with a Variety ofMicroorganisms

DHN is inexpensive and readily available commercially. We found thatnovel glycosides of it could be prepared by standard means. Thus theprotected DHN-β-D-galactopyranoside (7) was made by couplingacetobromogalactose with DHN in acetone-water in the presence of sodiumhydroxide. After work-up, deprotection in methanol containing acatalytic quantity of sodium methoxide furnished the desiredDHN-β-D-galactopyranoside (8) as an off-white solid. Details of thesynthesis are given below in syntheses 7 and 8.

This compound was tested on agar plates by multi-point inoculation. Theplates were made from Columbia agar (Oxoid, Basingstoke, UK) (100 ml)supplemented with ferric ammonium citrate (FAC) (50 mg). After the agarhad been autoclaved (121° C.) and cooled to 50° C., a filter sterilizedsolution of the substrate (8) (30 mg) dissolved in N-methylpyrrolidone(NMP) (200 μL) was added, thus giving a substrate concentration of 300mg/L. After pouring, the plates were inoculated with a range ofmicroorganisms and incubated at 37° C. for 18 h in air (Table 1). Thoseorganisms positive for β-galactosidase could be easily distinguishedfrom those negative for this enzyme by the development of a strongpurple-maroon colour in the former.

TABLE 1 DHN-β-D- galactopyranoside 8 Organism Colour 1 Escherichia coliNCTC 10418 ++ 2 Klebsiella pneumoniae NCTC 9528 ++ 3 Providenciarettgeri NCTC 7475 − 4 Enterobacter cloacae NCTC 11936 ++ 5 Serratiamarcescens NCTC 10211 + 6 Salmonella typhimurium NCTC 74 − 7 Pseudomonasaeruginosa NCTC 10662 − 8 Yersinia enterocolitica NCTC 11176 − 9Burkholderia cepacia NCTC 10931 − 10 Acinetobacter baumannii NCTC 19606− 11 Steptococcus pyogenes NCTC 8306 − 12 Staphylococcus aureus (MRSA)NCTC 11939 − 13 Staphylococcus aureus NCTC 6571 − 14 Staphylococcusepidermidis NCTC 11047 − 15 Listeria monocytogenes NCTC 11994 − 16Enterococcus faecium NCTC 7171 Tr. 17 Enterococcus faecalis NCTC 775 −18 Bacillus subtilis NCTC 9372 − 19 Candida albicans ATCC 90028 − 20Candida glabrata NCPF 3943 −

Unless indicated otherwise, the symbols in each table have the followingmeaning; ++ means strong colour; + means less colour than ++; +/− meansless colour than +; Tr means a trace of colour, less than +/−; − meansno colour. The substrate (8) showed no obvious toxicity with any of theGram-positive or Gram-negative strains tested. Essentially identicalresults were obtained if other iron salts (e.g. iron(II) gluconate,iron(II) acetate, iron(II) citrate, iron(II) acetylacetonate, andiron(III) acetylacetonate) were substituted in place of FAC. The ironcompound can be either iron(II) (i.e. ferrous) or iron(III) (i.e.ferric). Both types of compound work well. The coloured chelate wasstill formed when the plates were incubated under anaerobic conditions,which is another useful feature of the present invention. It will beappreciated that the media and the reagents will all contain at leasttraces of iron compounds. However, the invention does not work unlessthe medium is supplemented with a sufficient amount of an iron compound.Concentrations of 200-600 mg/L of iron compound were found to besatisfactory. In contrast, supplementing the growth media with compoundsof other metals gives either no coloured endpoint or an extremely poorone to visualise. In an alternative to the above procedure, thesubstrate may be added to the agar prior to being autoclaved (121° C.for 20 minutes) with no decrease in sensitivity. The colour generated byDHN in combination with an iron salt is of about the same intensity asthat previously demonstrated by the reaction between1,2-dihydroxybenzene (catechol) and an iron salt at the sameconcentration [M. Burton, EP1438423, (2007)]. However, the colours aredifferent (the catechol-iron complex is black in solution) and this maybe an advantage in designing a particular test medium.DHN-β-D-galactopyranoside (8) may find application as an alternative tothe widely used enzyme substrate ONPG.

Example 2—Other DHN Substrates and Cleavage by Microorganisms with aRange of Enzymatic Marker Activities

Several other DHN substrates, intended for some of the most frequentlyencountered hydrolase activities in diagnostic microbiology, weresynthesised by the methods described below. These were the glycosidesβ-D-glucopyranoside (4), α-D-glucopyranoside (6), α-D-galactopyranoside(10) and N-acetyl-β-D-glucosaminide (15), the esterase substrateDHN-dicaprylate (16) and the phosphatase substrate, DHN-phosphatedisodium salt (17). DHN-β-D-ribofuranoside (2) was also made. Whenevaluated in the same manner as DHN-β-D-galactopyranoside (8), ingeneral these substrates were hydrolysed according to the known enzymeprofiles of the test strains (Tables 2 and 3). However,DHN-β-D-ribofuranoside (2) (Table 2) was of particular interest.

TABLE 2 DHN-β-D- DHN-α-D- DHN-α-D- DHN-N-acetyl-β-D- ribofuranoside 2galactopyranoside 10 glucopyranoside 6 glucosaminide 15 Organism ColourColour Colour Colour 1 Escherichia coli NCTC 10418 ++ Tr. + − 2 Serratiamarcescens NCTC 10211 ++ − ++ ++ 3 Pseudomonas aeruginosa NCTC 10662 − −− − 4 Burkholderia cepacia 1222 − − − − 5 Yersinia enterocolitica NCTC11176 − − Tr. + 6 Salmonella typhimurium NCTC 74 ++ − − − 7 Citrobacterfreundii NCTC 9750 or 46262 ++ − − − 8 Morganella morganii 462403 (wild)++ − − − 9 Enterobacter cloacae NCTC 11936 ++ Tr. ++ +/− 10 Providenciarettgeri NCTC 7475 ++ − − − 11 Bacillus subtilis NCTC 9372 − − ++ − 12Enterococcus faecails NCTC 775 − − ++ ++ 13 Enterococcus faecium NCTC7171 − − Tr. ++ 14 Staphylococcus epidermidis NCTC 11047 − − + − 15Staphylococcus aureus NCTC 6571 + − +/− − 16 MRSA NCTC 11939 + − +/− −17 Steptococcus pyogenes NCTC 8306 − − ++ ++ 18 Listeria monocytogenesNCTC 11994 − − ++ ++ 19 Candida albicans ATCC 90028 − − − − 20 Candidaglabrata NCPF 3943 − − − −

TABLE 3 DHN- DHN-β-D- DHN-phosphate dicaprylate 16 ghicopyranoside 4disodium salt 17 Organism Colour Colour Colour 1 Escherichia coli NCTC10418 − Tr. +/− 2 Klebsiella pneumoniae NCTC 9528 − ++ ++ 3 Providenciarettgeri NCTC 7475 + ++ + 4 Enterobacter cloacae NCTC 11936 − + +/− 5Serratia marcescens NCTC 10211 + ++ ++ 6 Salmonella typhimurium NCTC 74− − ++ 7 Pseudomonas aeruginosa NCTC 10662 + − − 8 Yersiniaenterocolitica NCTC 11176 − − +/− 9 Burkholderia cepacia NCTC 10931 − −+/− 10 Acinetobacter baumannii NCTC 19606 + − − 11 Steptococcus pyogenesNCTC 8306 − − − 12 Staphylococcus aureus (MRSA) NCTC 11939 − − + 13Staphylococcus aureus NCTC 6571 − − + 14 Staphylococcus epidermidis NCTC11047 − − − 15 Listeria monocytogenes NCTC 11994 − + + 16 Enterococcusfaecium NCTC 7171 − + − 17 Enterococcus faecalis NCTC 775 − + − 18Bacillus subtilis NCTC 9372 − + − 19 Candida albicans ATCC 90028 − − −20 Candida glabrata NCPF 3943 − − −

When challenged with three strains of staphylococci, only the S. aureusstrains NCTC 6571 and MRSA NCTC 11939 were able to hydrolyse it. It wasunaffected by S. epidermidis NCTC 11047. This strongly suggests thatDHN-β-D-ribofuranoside (2) has potential utility in the detection ofMRSA and is able to differentiate this organism from other species ofstaphylococci that can cause interference in its positiveidentification. Other chromogenic β-D-ribofuranosides have already beenevaluated for this purpose [M. Burton, EP1438424, (2006)]. Theunsubstituted DHN-glycosides tested in the present invention were allprepared by means that have been reported in the literature for othermono- or di-phenolic aglycones. The various glycosyl donors employedwere all readily prepared intermediates such as acetohalosugars ortrichloroacetimidates. One disadvantage of DHN enzyme substrates in agarplate media is that the coloured iron-complex diffuses, as occurs alsowith substrates made from 1,2-diydroxybenzene or esculetin. Thediffusion is no greater than that which occurs with esculin (theβ-D-glucopyranoside of esculetin). As esculin is used commercially inagar plate media, substrates derived from DHN may also find applicationin agar plate media notwithstanding their capacity to diffuse. However,it would seem that they are much more advantageously employed in liquidbroth media or in agar tube media.

Example 3—DHN-β-D-Glucuronide Substrates and their Testing on VariousMicroorganisms

Tube or liquid media containing the fluorogenic compound MUG(4-methylumbelliferyl β-D-glucuronide) are extensively used to detect E.coli. Often these systems also contain the substrate ONPG for thedetection of β-D-galactosidase activity and therefore total coliforms.The media Colitag® (CPI International, Santa Rosa, USA) and ColiLert®(Idexx Laboratories, Westbrook, USA) both utilise MUG plus ONPG fordetecting E. coli and total coliforms. The disadvantage of MUG is that aUV source is required to visualise the fluorescence associated with E.coli. It would be an advantage to have a chromogenic glucuronide thatcan detect E. coli with incident visible light. Accordingly, the novelcompound DHN-β-D-glucuronide (12) was prepared in both itscyclohexylammonium salt form (12a) (hereinafter referred to as the CHAsalt) and its sodium salt form (12b). In the preliminary evaluationagainst twenty different microorganisms on agar plates containing FAC,both salt forms of the DHN-β-D-glucuronide (12a and 12b) were hydrolysedequally well by E. coli (as judged by the strong colour produced witheach) (Table 4). Just as significantly, E. coli was the only speciesable to effect hydrolysis; the other 19 strains were allβ-D-glucuronidase negative.

TABLE 4 DHN-β-D-glucuronide DHN-β-D-glucuronide CHA salt 12a sodium salt12b Organism Colour Colour 1 Escherichia coli NCTC 10418 ++ ++ 2Serratia marcescens NCTC 10211 − − 3 Pseudomonas aeruginosa NCTC 10662 −− 4 Burkholderia cepacia 1222 − − 5 Yersinia enterocolitica NCTC 11176 −− 6 Salmonella typhimurium NCTC 74 − − 7 Citrobacter freundii NCTC 9750or 46262 − − 8 Morganella morganii 462403 (wild) − − 9 Enterobactercloacae NCTC 11936 − − 10 Providencia rettgeri NCTC 7475 − − 11 Bacillussubtilis NCTC 9372 − − 12 Enterococcus faecails NCTC 775 − − 13Enterococcus faecium NCTC 7171 − − 14 Staphylococcus epidermidis NCTC11047 − − 15 Staphylococcus aureus NCTC 6571 − − 16 MRSA NCTC 11939 − −17 Steptococcus pyogenes NCTC 8306 − − 18 Listeria monocytogenes NCTC11994 − − 19 Candida albicans ATCC 90028 − − 20 Candida glabrata NCPF3943 − −

Example 4—Sensitivity of DHN-β-D-Glucuronide to Various E. coli IsolatesCompared to Standard Chromogenic Indoxyl Glucuronide Substrates

In order to obtain a fuller picture of the sensitivity ofDHN-β-D-glucuronide (12a) it was screened with 100 different clinicalisolates of E. coli in a liquid medium containing FAC. The isolates werechosen at random from the Microbiology Department, Freeman Hospital,Newcastle Upon Tyne, UK. The effectiveness of DHN-β-D-glucuronide (12a)was compared with three other media which all containedindoxyl-β-D-glucuronides. One was a commercial medium, CPS ID 3(bioMérieux SA, Lyon, France). CPS ID 3 contains complementarychromogenic substrates; Rose-β-D-glucuronide (6-chloro-3-indolylβ-D-glucuronide) of undisclosed salt form [at 250 mg/L] for thedetection of β-D-glucuronidase activity (producing red or pink colonies)and X-β-D-glucoside (5-bromo-4-chloro-3-indolyl β-D-glucopyranoside) [50mg/L] for the detection of β-D-glucosidase (producing green colonies)[M. Casse et al, U.S. Pat. No. 8,216,802 (2012)]. This medium wasemployed as a control. Among the β-D-glucuronidase producing strains ofE. coli there is a large variation in the quantity of the enzymeproduced and it is almost certain that the CPS ID 3 media has beenrigorously optimised to allow good growth of all the target organismsand maximum expression of the target enzymes. Therefore, two indoxylglucuronides, X-β-D-glucuronide CHA salt and Rose-β-D-glucuronide CHAsalt were also tested in a simple agar medium to allow a directcomparison of the sensitivity of these indoxyl glucuronides when used ina medium that has not been optimised for the growth of the targetorganism. The comparison of the results for Rose-β-glucuronide and theCPS ID 3 medium was of particular significance as the commercial mediumalso uses Rose-β-glucuronide for the detection of E. coli. Forconsistency, the three glucuronides were all chosen as their CHA salts.As already stated, is not anticipated that the salt form is critical totheir performance. Currently, X-β-D-glucuronide is often used as eitherthe CHA salt or the sodium salt. Brenner and colleagues [K. P. Brenneret al, Appl. Environ. Microbiol., 59, 3534-3544, (1993)] found nodifference in the performance of the CHA and sodium salt forms withtheir application using indoxyl-β-D-glucuronide, neither in respect ofcolour development nor in the recovery of E. coli.

The two indoxyl glucuronides used produce insoluble endpoints followinghydrolysis, as does the CPS ID 3 medium. It was therefore necessary totest these two substrates on agar plates. In contrast,DHN-β-D-glucuronide (12) gives a much more soluble endpoint best suitedto liquid media and was therefore tested in a broth medium. The brothwas prepared using proteose peptone (2 g), NaCl (1 g) and FAC (100 mg)in DI water (180 mL). This mixture was autoclaved and cooled to roomtemperature before being dispensed into bijoux (100×1.8 mL).DHN-β-D-glucuronide CHA salt (12a) (60 mg) was dissolved in water (20mL) and filtered to sterilize before being aseptically dispensed intosuccessive bijoux (0.2 mL) containing the broth solution. Thebroth/substrate solutions were then inoculated with bacterialsuspensions made up to 0.5 McFarland standard (2 μL per bijoux).X-β-D-glucuronide CHA salt (Glycosynth Ltd, Warrington, UK) (10 mg) wasdissolved in NMP (200 μL). Rose-β-D-glucuronide CHA salt (GlycosynthLtd, Warrington, UK) (20 mg) was dissolved in NMP (200 μL). Thesesolutions were then added to Columbia agar (Oxoid, Basingstoke, UK) (100mL) and inoculated with bacterial suspensions made up to 0.5 McFarlandstandard (1 μL). The strains of E. coli used are listed in table 5. Theplates and broths were incubated at 37° C. for 18 hours in air. Thegreen colonies seen on the CPS ID 3 media were indicative ofβ-D-glucosidase activity.

TABLE 5 X-β-D-glucuronide Rose-β-D-glucuronide DHN-β-D-glucuronide RefOrganism Reference CPS ID 3 agar CHA salt CHA salt CHA salt 12a broth 1E. coli 260471B +/−Red − − − 2 E. coli 260464G Red Green Red Purple 3 E.coli 260481J Red Green Red Purple 4 E. coli 260480M Red Green Red Purple5 E. coli 260521S Red Green Red Purple 6 E. coli 260578D +/−Red − − − 7E. coli 260537E Red − − − 8 E. coli 260522Z Red Green Red Purple 9 E.coli 260541R +/−Red Green Red Purple 10 E. coli 260538H Red − − − 11 E.coli 260539Y Red Green Red Purple 12 E. coli 260545G Red Green RedPurple 13 E. coli 260459W Red Green Red Purple 14 E. coli 260458Y RedGreen Red Purple 15 E. coli 260441G Red Green Red Purple 16 E. coli260440S Red Green Red Purple 17 E. coli 260508Y Red Green Red Purple 18E. coli 260504N Red Green Red Purple 19 E. coli 260503Z Red Green RedPurple 20 E. coli 260554D Red Green Red Purple 21 E. coli 260502G RedGreen Red Purple 22 E. coli 260532S Red Green Red Purple 23 E. coli260533G Red Green Red Purple 24 E. coli 260536Q Red Green Red Purple 25E. coli 260548Q Red Green Red Purple 26 E. coli 260549E Red Green RedPurple 27 E. coli 260553X − − − − 28 E. coli 260547N Red Green RedPurple 29 E. coli 260563B Red Green Red Purple 30 E. coli 260564R RedGreen Red Purple 31 E. coli 260555L Red Green Red Purple 32 E. coli260511X Red Green Red Purple 33 E. coli 260515Z Red − Red Purple 34 E.coli 260514G Red − − − 35 E. coli 260505Q Red − − − 36 E. coli 260510RRed Green Red Purple 37 E. coli 260364H Tr. Red − − − 38 E. coli 260406YRed Green Red Purple 39 E. coli 260492J Red Green Red Purple 40 E. coli260486L Red Green Red Purple 41 E. coli 260485D Red Green Red Purple 42E. coli 260479Q Red Green Red Purple 42 E. coli 260506E Red Green RedPurple 44 E. coli 260478N Tr. Red − − − 45 E. coli 260463D Red Green RedPurple 46 E. coli 260396Z Red Green Red Purple 47 E. coli 260370C RedGreen Red Purple 48 E. coli 260262P Red Green Red Purple 49 E. coli260280Z Red Green Red Purple 50 E. coli 260375E Red Green Red Purple 51E. coli 260400N − − − − 52 E. coli 260404W Red Green Red Purple 53 E.coli 260401Q Red Green Red Purple 54 E. coli 260402H Red Green RedPurple 55 E. coli 260411Z Red Green Red Purple 56 E. coli 260407F RedGreen Red Purple 57 E. coli 260408C Red Green Red Purple 58 E. coli260509W Red Green Red Purple 59 E. coli 260433H Red Green Red Purple 60E. coli 260432E Red Green Red Purple 61 E. coli 260431N Red Green RedPurple 62 E. coli 260428T Red Green Red Purple 63 E. coli 260426F RedGreen Red Purple 64 E. coli 260425A Red Green Red Purple 65 E. coli260483R − − − − 66 E. coli 260494B Red Green Red Purple 67 E. coli260495R − − − Purple 68 E. coli 260439C Red Green Red Purple 69 E. coli260438F Red Green Red Purple 70 E. coli 260437A Red − Tr. Red − 71 E.coli 260412Q Red Green Red Purple 72 E. coli 260497D Red Green RedPurple 73 E. coli 260416W Red − − Purple 74 E. coli 260417P Red GreenRed Purple 75 E. coli 260422Y Red Green Red Purple 76 E. coli 260406ARed − − − 77 E. coli 260405P Red Green Red Purple 78 E. coli 260399E RedGreen Red Purple 79 E. coli 260435W Red Green Red Purple 80 E. coli260434Y Red Green Red Purple 81 E. coli 2603121P Tr. Red − − − 82 E.coli 260310H Red Green Red Purple 83 E. coli 260436P Red Green RedPurple 84 E. coli 260398Q Red Green Red Purple 85 E. coli 260345F RedGreen Red Purple 86 E. coli 260390B − − − − 87 E. coli 260414H Red GreenRed Purple 88 E. coli 260415Y Red Green Red Purple 89 E. coli 260313ATr. Red − − − 90 E. coli 260316T − − − − 91 E. coli 260424P Red GreenRed Purple 92 E. coli 260348K Red Green Red Purple 97 E. coli 260354WRed Green Red Purple 98 E. coli 260333P − − − − 99 E. coli 260330Y RedGreen Red Purple 100 E. coli 260327V Red Green Red Purple 101 E. cloacae260329R Tr. Green − − − 102 E. cloacae NCTC 11936 Tr. Green − − − 103 E.faecium NCTC 7171 Green − − − 104 E. faecalis NCTC 775 Green − − − 105E. coli O157 non- − − − − toxigenic Red, Green, Black or Purple meanstrong colour; +/−means less colour than strong; Tr means a trace ofcolour, less than +/−; − means no colour.

The results of table 5 (lines 1-100) are summarised below (Table 6) forthe 100 E. coli strains after 18 h incubation;

TABLE 6 Negative Positive Substrate/Medium strains strains % SensitivityCPS ID 3 7 93 93 DHN-β-D-glucuronide CHA salt 18 82 82 12a brothRose-β-D-glucuronide CHA salt 19 81 81 X β-D-glucuronide CHA salt 21 7979

The commercial medium, CPS ID3, was the most sensitive with 93/100 of E.coli strains giving red colonies. The excellent performance of thismedium was to be expected, as it most probably contains inducers ofβ-D-glucuronidase activity and/or optimal conditions for the expressionof this enzyme. That not all strains were detected by this medium isunderstandable, as a small percentage of all E. coli strains is negativefor β-D-glucuronidase. Surprisingly, the next most sensitive medium wasDHN-β-D-glucuronide (12a) [purple solutions] 82/100 strains. The novelsubstrate showed higher sensitivity than Rose-β-D-glucuronide [redcolonies] (81/100 strains) when it was used in the simple Columbia agarmedium. Considering that Rose-β-D-glucuronide is the same substrate asemployed in the CPS ID3 medium, it shows how those skilled in the artcan develop a medium to increase the sensitivity of the substrate whenchallenged with many different strains of microorganisms.X-β-D-glucuronide [green colonies] gave the lowest sensitivity (79/100strains) in the simple agar medium, yet this substrate is currently veryextensively used in commercial media to detect E. coli. More surprisingstill, DHN-β-D-glucuronide (12a) visualised one strain (E. coli 260495R)that was not detected by CPS ID3 or by the other two media containingthe indoxyl glucuronides. In addition to the 100 strains of E. coli, allfour media were tested with four other stains of Enterobacteriaceaeknown to be β-D-glucuronidase-negative, as well as oneβ-D-glucuronidase-negative strain of E. coli (E. coli 0157non-toxigenic) (Table 5, lines 101-105). All five of these strains werenegative on all the media, thus showing 100% specificity forβ-D-glucuronidase-producing E. coli over these other organisms.

Example 5—Combination of DNH-β-D-Glucuronide and ONPG to Simulate a DualChromogenic Systems for Distinguishing Galactosidase and GlucuronidasePositive/Negative Microorganisms

Because many E. coli produce both β-D-glucuronidase andβ-D-galactosidase, DHN-β-D-glucuronide (12a) was tested as in the brothmedium described above but in the presence of ONPG (at a concentrationof 1.5 g/L). This was done to see if the colour produced by the ironcomplex could mask the yellow of o-nitrophenol. This would be essentialto successfully visualise any E. coli in a dual-chromogenic (or possiblymulti-chromogenic) system. It was found that E. coli expressing bothβ-D-glucuronidase and β-D-galactosidase now gave purple-brown solutionswith DHN-β-D-glucuronide (12a). The colour of these solutions of mixedchromogens could be very readily distinguished with the unaided eye fromthe yellow colour of those strains, such as E. cloacae, that producedβ-D-galactosidase only. Similarly, a combination of DHN-β-D-glucuronide(12a) with ONPG was able to detect the β-D-glucuronidase activity ofShigella sonnei, an important pathogen that is generally positive forboth β-D-glucuronidase and β-D-galactosidase. The above results clearlydemonstrate the potential of DHN-β-D-glucuronide (12) to be used in aliquid medium to detect E. coli, either on its own or in combinationwith other substrates (e.g. with ONPG). Although the strains tested wereof clinical origin, it will be appreciated that DHN-β-D-glucuronide (12)of the present invention may be used to screen samples from food,environmental sources and water. Moreover, those skilled in the art ofdeveloping such media could add other ingredients, such as specificenzyme or growth inducers, enzyme or growth inhibitors or othermetabolic regulators to ensure the optimum performance of the substrateswithin the media. Inducers of β-D-glucuronidase in an enzymatic methodfor detecting E. coli have been disclosed [H. Nelis, U.S. Pat. No.5,861,270, (1999)] and a commercially produced enzyme inducer cocktailhas been used in a test for the enumeration of E. coli in drinking water[S. O. Van Poucke and H. J. Nelis, J. Appl. Microbiol., 89, 390-396,(2000)]. In the method of Monget et al for identifying E. coli frombiological samples [D. Monget et al, U.S. Pat. No. 8,334,112 (2012)]glucuronate and methyl-β-glucuronide are cited as the preferred inducersof β-glucuronidase. DHN-β-D-glucuronide-6′-methyl ester (13) was alsohydrolysed by β-D-glucuronidase-positive strains, but the colour wasweaker than with (12a) or (12b).

Example 6—Nitrated DHN-Substrate

Having established the utility of DHN as a useful core molecule as abase for chromogenic enzyme substrates, we sought to address the issueof diffusion of the core molecule that would limit its application in oron solid plate media, such as agar media. A possible way to reduce thesolubility of a core molecule is to increase its molecular weight orsize. Nitration of the fully protected DHN-β-D-ribofuranoside (1)introduced a nitro group at the 1-position of the DHN nucleus. Themethod is described in detail below. Deprotection afforded1-nitro-DHN-β-D-ribofuranoside (19), and this was tested on agar plateswith FAC in exactly the same manner as the unsubstituted compound (2).Unfortunately, the 1-nitro-DHN-iron complex still diffused extensively.However, the colour of this chelate was more distinctly red than theDHN-iron complex, so compounds of this type may be preferred over theunsubstituted-DHN derivatives depending on the requirements.

Example 7—Halogenated DHN-Substrates

Halogenation of DHN was then explored. Bromination of DHN was carriedout by the method of Zincke and Fries [T. Zincke and K. Fries, Annalen,334, 365, (1904)]. Using a ratio of 4 mol of molecular bromine to 1 molof DHN in acetic acid as described by these authors and as detailedbelow gave the anticipated 1,4-dibromo-DHN in good yield. By increasingthe amount of bromine to 8 mol, (again as described by Zincke andFries), the expected 1,4,6,7-tetrabromo-DHN (20) was also obtained ingood yield. Both these brominated derivatives were glycosylated as the3-D-ribofuranosides, after which the substrates were separatelyincorporated into agar plates containing FAC for microbiologicalevaluation using a range of organisms previously tried with theunsubstituted DHN-glycosides. The results with both1,4-dibromo-DHN-β-D-ribofuranoside and1,4,6,7-tetrabromo-DHN-β-D-ribofuranoside were equally disappointing;both gave a very strong background colouration to the whole plate makingit impractical to see any positive reactions.

In the publication of Zincke and Fries, the authors reported thattreatment of 1,4,6,7-tetrabromo-DHN (20) with tin (II) chloride led tothe removal of the bromines in the 1 and 4 positions. Their work wasrepeated and the expected 6,7-dibromo-DHN (21) was obtained smoothly andin good yield (68%). 6,7-Dibromo-DHN (21) is isomeric with the1,4-dibromo compound which had proven unsuccessful as an aglycone forthe detection of microorganisms. Notwithstanding this latter fact, weproceeded to evaluate the properties of 6,7-dibromo-DHN (17) as anaglycone for artificial chromogenic enzyme substrates. Firstly we foundthat this molecule was efficiently converted into6,7-dibromo-DHN-β-D-ribofuranoside (23).6,7-Dibromo-DHN-β-D-ribofuranoside (23) was incorporated into Columbiaagar plates containing FAC (500 mg/L) at a substrate concentration of300 mg/L. The plates were made as described for DHNβ-D-galactopyranoside (8), except that the substrate was dissolved inDMSO (200 μL). After inoculation and incubation for 18 h at 37° C. inair, 6,7-dibromo-DHN-β-D-ribofuranoside (23) produced largely discretered-brown or maroon colonies upon hydrolysis by a number of differentGram-negative bacteria (Table 7). There was little or no diffusion ofthe colour into the surrounding medium and the background colouration ofthe plates was minimal. The growth of Gram-positive bacteria was mainlysupressed by this substrate.

TABLE 7 6,7-Dibromo- 6.7-Dibromo- 6,7-Dibromo- 6,7-Dibromo- DHN-β-D-DHN-β-D- DHN-β-D- DHN-β-D- glucuronide ribofuranoside 23galactopyranoside 25 glucopyranoside 27 CHA salt 29 Strains A ColourColour Colour Colour 1 Escherichia coli NCTC 10418 − ++ Tr. ++ 2Serratia marcescens NCTC 10211 ++ + ++ − 3 Pseudomonas aeruginosa NCTC10662 + − − − 4 Burkholderia cepacia 1222 +/− +/− + − 5 Yersiniaenterocolitica NCTC 11176 − − − − 6 Salmonella typhimurium NCTC 74 ++ −− − 7 Citrobacter freundii NCTC 9750 or 46262 ++ + + − 8 Morganellamorganii 462403 (wild) ++ − − − 9 Enterobacter cloacae NCTC 11936 ++ − −− 10 Providencia rettgeri NCTC 7475 − − ++ − 11 Bacillus subtilis NCTC9372 − − + − 12 Enterococcus faecails NCTC 775 − Tr. Tr. − 13Enterococcus faecium NCTC 7171 − +/− +/− − 14 Staphylococcus epidermidisNCTC 11047 − − − − 15 Staphylococcus aureus NCTC 6571 − − Tr. − 16 MRSANCTC 11939 − − Tr. − 17 Steptococcus pyogenes NCTC 8306 − − − − 18Listeria monocytogenes NCTC 11994 − +/− +/− − 19 Candida albicans ATCC90028 − − − − 20 Candida glabrata NCPF 3943 − − − −

Example 8—Further Halogenated DHN-Glycosides

Additional glycosides based on 6,7-dibromo-DHN were also produced toshow the generality of this aspect of the invention (Table 7). Thesynthetic methods are described in detail in the section below. Theβ-D-galactopyranoside (25), the β-D-glucopyranoside (27) and theβ-D-glucuronide CHA salt (29) all gave non-diffuse colonies on agarplates. Gram-negative species grew well but the growth of someGram-positive organisms was adversely affected by the substrates. Theseresults showed that the 6,7-dibromo-DHN (21) is a suitable chromogeniccore molecule for substrates incorporated into agar plates for thedetection of Gram-negative organisms. Therefore, from a singleinexpensive core molecule nucleus, i.e., DHN, we have succeeded inproducing workable chromogenic enzyme substrates that are suitable forinclusion in either liquid or solid microbiological growth media. Itwill be appreciated by those skilled in the art that media fallingbetween the two extremes of liquid and solid may be chosen depending onthe specific application.

Example 9—Substrates Derived from DHN-6-Sulfonic Acid

DHN-6-sulfonic acid is also commercially available, most commonly as itssodium salt. As this is an unsymmetrical molecule, mono-glycosylation(or mono-derivatisation) of the aromatic ring hydroxyl groups is capableof yielding two different products. Prior to the present invention,glycosides of DHN-6-sulfonic acid were unknown. We succeeded in couplingacetobromogalactose to DHN-6-sulfonic acid by means of a phase-transferreaction with tetrabutylammonium bromide as the catalyst. This gave amixture of the protected di-galactoside and a protected mono-galactoside(30). The mono-galactoside (30) was isolated by column chromatography.Deprotection with sodium methoxide gave a DHN-6-sulfonicacid-β-D-galactopyranoside sodium salt (31). When tested in Columbiaagar plates containing FAC (500 mg/L) at a substrate concentration of300 mg/L it produced purple to maroon colonies after hydrolysis (Table8). The colour was slightly different to that obtained byDHN-β-D-galactopyranoside (8) under the same conditions, but the colourwas equally diffuse on agar plates.

TABLE 8 DHN-6-sulfonic acid-β-D- DHN-6-sufonic galactopyranosideacid-phosphate sodium salt 31 trisodium salt 32 Organism Colour Colour 1Escherichia coli NCTC 10418 + − 2 Klebsiella pneumoniae NCTC 9528 − Tr.3 Providencia rettgeri NCTC 7475 + Tr. 4 Enterobacter cloacae NCTC11936 + − 5 Serratia marcescens NCTC 10211 − +/− 6 Salmonellatyphimurium NCTC 74 − Tr. 7 Pseudomonas aeruginosa NCTC 10662 − − 8Yersinia enterocolitica NCTC 11176 − − 9 Burkholderia cepacia NCTC 10931− − 10 Acinetobacter baumannii NCTC 19606 − − 11 Steptococcus pyogenesNCTC 8306 − − 12 Staphylococcus aureus (MRSA) NCTC 11939 − Tr. 13Staphylococcus aureus NCTC 6571 − Tr. 14 Staphylococcus epidermidis NCTC11047 − − 15 Listeria monocytogenes NCTC 11994 − − 16 Enterococcusfaecium NCTC 7171 − − 17 Enterococcus faecalis NCTC 775 − − 18 Bacillussubtilis NCTC 9372 − − 19 Candida albicans ATCC 90028 − − 20 Candidaglabrata NCPF 3943 − −

However, the DHN-6-sulfonic acid-β-D-galactopyranoside sodium salt (31)has the benefit of being very soluble in aqueous media. It readilydissolved in water at ambient temperature (30 mg of substrate dissolvedin 1 mL DI water) without the need to add any polar solvents. This is apractical advantage over other types of artificial chromogenic enzymesubstrates because the polar solvents normally employed to aid solution,like DMSO, exhibit toxicity to microorganisms. DHN-6-sulfonicacid-phosphate trisodium salt (32) was also prepared and tested on agarplates at a concentration of 300 mg/L with FAC (500 mg/L) (Table 8). Aswith compound 31, it was very soluble water and its real value is as asubstrate in liquid media.

Synthetic Methods

Materials

The glycosyl donors were all prepared by literature procedures. Allother reagents and solvents were purchased from Sigma-Aldrich(Gillingham, UK), Alfa Aesar (Heysham, UK) or Univar (Widnes, UK) exceptwhere stated differently. Flash column chromatography was performed onsilica gel C₆₀ (Fluorochem, Derbyshire, UK). TLC was carried out usingpre-coated silica plates (0.2 mm, UV₂₅₄). These were developed using UVfluorescence at 254 nm and 366 nm followed by spraying with H₂SO₄/MeOHand heating. Mixed solvent compositions are reported as volumetricratios. NMR spectra were recorded on a 270 MHz Joel NMR spectrometer (at270 MHz for ¹H and 68 MHz for ¹³C) or NMR spectra were recorded on a 400MHz Joel NMR spectrometer (at 400 MHz for ¹H and 100 MHz for ¹³C). Allchemical shifts are quoted in ppm relative to TMS. Optical rotationswere measured on an Optical Activity AA10 polarimeter. Melting pointswere determined with an Electrothermal A19200 apparatus and areuncorrected. All melting points are quoted to the nearest 0.5° C. HighResolution Mass Spectroscopy (HRMS) data were obtained using the EPSRCmass spectrometry service centre (Swansea, UK).

Synthesis 1 (Reference). DHN-2′,3′,5′-tri-O-acetyl-β-D-ribofuranoside(1)

DHN (14.9 g) was stirred in dichloromethane (DCM) (200 mL). BF₃.etherate(3 mL) was added to the mixture followed by2,3,5-tri-O-acetyl-D-ribofuranosyl-trichloroacetimidate [I. Chiu-Machadoet al, J. Carb. Chem., 14, 551, (1995)] (13 g) in DCM (100 mL). Afterapprox. 5 minutes the reaction mixture was poured into sat. aq. NaHCO₃solution (300 mL) and DCM (200 mL). The DCM layer was separated andwashed with sat. aq. NaHCO₃ solution (4×500 mL). TLC showed the reactionmixture still contained a large amount of unreacted DHN, therefore itwas washed with sat. sodium carbonate (2×500 mL). TLC then showed noremaining free DHN. The DCM layer was washed with water (500 mL) beforebeing dried over MgSO₄ and concentrated under reduced pressure toproduce an amber foam. The isolated foam was triturated in MeOH (50 mL)and the resultant white solid harvested by filtration to give compound 1(6.24 g, 54%). m.p. 142-144° C. [α]_(D) ²² −69° (c 0.99 in acetone).HRMS (ESI) for C₂₁H₂₆O₉N [M+NH₄]⁺: m/z calcd 436.1602; measured:436.1609.

Synthesis 2. DHN-β-D-ribofuranoside (2)

Compound 1 (2 g) was suspended in MeOH (6 mL), NaOMe solution in MeOH(2.17 M, 2.0 mL) was added and the reaction mixture was left at +4° C.overnight. TLC showed complete deprotection. The solution wasneutralised using AcOH (0.5 mL) and the solution concentrated underreduced pressure to give a white foam. The white foam was dissolved inIMS (15 mL) and left at +4° C. overnight to crystallise. Filtrationisolated compound 2 as a white solid (542 mg, 39%). m.p. 181-182° C.[α]_(D) ²² −143° (c 0.55 in acetone/water 1:1 v/v). HRMS (ESI) forC₁₅H₁₆O₆Na [M+Na]⁺: m/z calcd 315.0839; measured: 315.0844.

Synthesis 3 (Reference).DHN-2′,3′,4′,6′-tetra-O-acetyl-β-D-glucopyranoside (3)

DHN (80.6 g, 503 mmol) and acetobromoglucose (172.6 g) were stirred inacetone (1.1 L). A solution of NaOH (20 g) in DI water (400 mL) wasadded in one portion. The clear orange solution was stirred overnight atroom temperature. The reaction mixture was concentrated under reducedpressure until all the acetone had distilled off whereupon a gum formedin the residue. The aqueous solution was decanted off from the creamcoloured gum. The gum was dissolved in DCM (1 L) and washed with sat.NaHCO₃ (4×1 L) and DI water (2×1 L) before being dried (MgSO₄) andconcentrated under reduced pressure to give a yellow oil. The yellow oilwas triturated in MeOH (100 mL) to give a solid that was collected byfiltration. The obtained solid (63 g) was recrystallised from boilingMeOH (1 L) using charcoal (20 g) to give compound 3 (37.17 g, 18%) as awhite fluffy solid. m.p. 155-156° C., [α]_(D) ²² −24° (c 0.6 inacetone). HRMS (ESI) for C₂₄H₃₀O₁₁N [M+NH₄]⁺: m/z calcd 481.1453;measured: 481.1448. The ¹H-NMR spectral data were consistent with thatfound in the literature.

Synthesis 4. DHN-β-D-glucopyranoside (4)

Compound 3 (1.5 g) was suspended in MeOH (4.5 mL) and deprotected by themethod used to make compound 2. This afforded compound 4 as a whitesolid (930 mg, 94%). m.p. >210° C. decomp, [α]_(D) ²² −100° (c 1.01 inwater). HRMS (ESI) for C₁₆H₂₂O₇N [M+NH₄]⁺: m/z calcd 340.1391; measured:340.1397. The ¹H-NMR spectral data were consistent with that found inthe literature.

Synthesis 5. (Reference)DHN-2′,3′,4′,6′-tetra-O-acetyl-α-D-glucopyranoside (5)

In a 500 mL round bottom flask, a mixture of DHN (21.8 g), HgBr₂ (17.3g) and HgCN₂ (12.3 g) were stirred together in MeCN (250 mL) with 3 Åmolecular sieves (10 g) for 10 min. Acetobromoglucose (56 g) was addedand the mixture stirred at room temperature overnight. TLC then showedno remaining acetobromoglucose. The reaction mixture was filteredthrough Celite®, washing through with DCM (250 mL). The filtrate waswashed with sat. NaHCO₃ (4×300 mL) and DI water (2×300 mL) before beingdried (MgSO₄) and concentrated under reduced pressure to give a palebrown foaming oil (73.92 g). The oil was purified by flashchromatography using C₆₀ silica gel (1 kg), eluting with toluene/acetone10:1 v/v, collecting fractions of 200 mL. Fractions 6-20 wereconcentrated under reduced pressure to produce an orange oil (30.71 g).The orange oil was triturated in IMS (50 mL) and the resultant solid(compound (3)) was collected by filtration and discarded. The filtratefrom the obtained solid was concentrated under reduced pressure to givea yellow oil which was triturated in IMS (150 mL) and left at +4° C.overnight. The resultant pale yellow solid was collected by filtration(6.45 g). Recrystallisation from IMS (30 mL) using charcoal (2 g) gavecompound 5 as an off-white solid (2.77 g). [α]_(D) ²⁶ +202° (c 0.42 inCHCl₃). The filtrate from the recrystallization was concentrated underreduced pressure to afford a second crop of compound 5 (1.9 g, 2.8%)[α]_(D) ²⁶ +213° (c 0.5 in CHCl₃), m.p. 130-130.5° C. HRMS (ESI) forC₂₄H₃₀O₁₁N [M+NH₄]⁺: m/z calcd 508.1813; measured: 508.1815.

Synthesis 6. DHN-α-D-glucopyranoside (6)

Compound 5 (1 g) was suspended in MeOH (3 mL) and deprotected by themethod used in Synthesis 2 to make compound 2 to give compound 6 as awhite fluffy solid (105 mg, 16%). m.p. 164-166° C., [α]_(D) ²⁶ +244° (c0.25 in water). HRMS (ESI) for C₁₆H₂₂O₇N [M+NH₄]⁺: m/z calcd 340.1391;measured: 340.1394.

Synthesis 7. DHN-2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranoside (7)

This compound was prepared from DHN (80.6 g) and acetobromogalactose(172.6 g) by the method used in Synthesis 3 to make compound 3. Thisgave compound 7 (31.5 g, 15%) as a white fluffy solid. m.p. 83-84° C.,[α]_(D) ²² +6° (c 1 in acetone). HRMS (ESI) for C₂₄H₃₀O₁₁N [M+NH₄]⁺: m/zcalcd 508.1813; measured: 508.1805.

Synthesis 8. DHN-β-D-galactopyranoside (8)

Compound 7 (1.5 g) was suspended in MeOH (4.5 mL) and deprotected by themethod used to make compound 2 (Synthesis 2) to afford compound 8 (385mg, 39%) as an off-white solid. m.p. >230° C. decomp, [α]_(D) ²² −87° (c0.62 in water). HRMS (ESI) for C₁₆H₂₂O₇N [M+NH₄]⁺: m/z calcd 340.1391;measured: 340.1397.

Synthesis 9 (Reference).DHN-2′,3′,4′,6′-tetra-O-acetyl-α-D-galactopyranoside (9)

Compound 9 was prepared from DHN and acetobromogalactose by the methodused to make the analogous glucopyranoside 5 (Synthesis 5). HRMS (ESI)for C₂₄H₃₀O₁₁N [M+NH₄]⁺: m/z calcd 508.1813; measured: 508.1814.

Synthesis 10. DHN-α-D-galactopyranoside (10)

Compound 9 (5 g) was suspended in MeOH (15 mL) and NaOMe solution inMeOH (2.17 M, 1.5 mL) was added. After overnight reaction mixture wasconcentrated under reduced pressure to give a brown foam. The obtainedfoam was purified by flash chromatography using C₆₀ silica gel (220 g),eluting with DCM/MeOH 15:1 v/v, collecting fractions of 50 mL. Fractions53-72 were combined and concentrated under reduced pressure to givecompound 10 as a pink foaming solid (1.2 g, 37.5%). [α]_(D) ²³ +196° (c0.23 in water).

Synthesis 11 (Reference).DHN-2′,3′,4′,-tri-O-acetyl-β-D-glucuronide-6′-methyl ester (11)

A mixture of DHN (34.4 g) and1,2,3,4-tetra-O-acetyl-β-D-glucuronide-6-methyl ester (MTAG) [G. N.Bollenback et al, J. Am. Chem. Soc., 77, 3310, (1955)] (40 g) was heatedin an oil bath to 120° C. on a rotary evaporator under reduced pressureuntil a homogeneous melt was obtained. PTSA (150 mg) in 1:1 v/vAcOH/Ac₂O (1 mL) was added and the mixture stirred at 120° C. on arotary evaporator under reduced pressure for 1 hour. TLC showed someremaining MTAG, therefore PTSA (150 mg) in 1:1 v/v AcOH/Ac₂O (1 mL) wasadded and the mixture stirred at 120° C. under reduced pressure for afurther 30 min. TLC then showed no remaining MTAG. The dark oil wasallowed to cool to room temperature overnight before being dissolved inDCM (300 mL). The solution was washed with sat. NaHCO₃ (4×50 mL), DIwater (500 mL) and brine (500 mL) before being dried (MgSO₄) andconcentrated under reduced pressure to give a brown foaming oil (59.1g). The foam was purified by flash chromatography using C₆₀ silica gel(1 Kg), eluting with toluene/acetone 10:1 v/v, collecting fractions of200 mL. Fractions 19-26 were combined and concentrated under reducedpressure to produce a red solid (29.66 g). The red solid was trituratedin IMS (150 mL) and left at +4° C. overnight to completecrystallisation. The resultant pale yellow fluffy solid was collected byfiltration to give compound 11 (12.6 g, 25%). m.p. 191-192° C., [α]_(D)²³ −26° (c 0.5 in CHCl₃. HRMS (ESI) for C₂₃H₂₈O₁₁N [M+NH₄]⁺: m/z calcd494.1657; measured: 494.1646.

Synthesis 12a. DHN-β-D-glucuronide CHA Salt (12a)

Compound 11 (6.1 g) was dissolved in acetone (75 mL). A solution of NaOH(2.81 g) in DI water (37.5 mL) was added. The mixture was stirred atroom temperature for 2 hours. TLC showed no remaining protectedmaterial. The solution was passed down an Amberlite®IR120 H⁺ ionexchange resin column (50 g). The eluent containing the product wasbasified using cyclohexylamine (5 mL). A white precipitate formed. Themixture was left at +4° C. overnight. The white fluffy solid wascollected by filtration, washing with DI water then acetone to givecompound 12a as a white fluffy solid (2.8 g, 53%). m.p. 223-224° C.,[α]_(D) ¹⁹ −96° (c 0.5 in water). HRMS (ESI) for O₁₆H₁₆O₈ [M+H]⁺: m/zcalcd 335.0772; measured: 335.0767.

Synthesis 12b. DHN-β-D-glucuronide sodium Salt (12b)

Compound 11 (1.56 g) was dissolved in acetone (21 mL). A solution ofNaOH (0.446 g) in DI water (1 mL) was added. The mixture was stirred atroom temperature overnight. TLC showed no remaining protected material.A brown precipitate had formed in the solution. This solid was collectedby filtration to give the desired compound 12b (1.16 g, 99%). m.p.53-55° C., [α]_(D) ²³ −25° (c 0.995 in water).

Synthesis 13. DHN-β-D-glucuronide-6′-methyl ester (13)

Compound 11 (1.0 g) was suspended in MeOH (3 mL) and NaOMe solution inMeOH (2.17 M, 0.6 mL) was added. The solid slowly dissolved and a creamprecipitate began to form. The mixture was neutralised to pH 6-7 usingAcOH (0.2 mL). The solid dissolved giving an orange solution which wasconcentrated under reduced pressure to give compound 13 as an orangefoam (849 mg) which appeared to contain about 15% inorganic salt.[α]_(D) ¹⁹ −100° (c 0.1 in water). HRMS (ESI) for C₁₇H₂₂O₈N [M+NH₄]⁺:m/z calcd 368.1340; measured: 368.1339.

Synthesis 14 (Reference).DHN-N-acetyl-3′,4′,6′-tri-O-acetyl-β-D-glucosaminide (14)

In a 250 mL round bottom flask a mixture of acetochloroglucosamine (30g) and DHN (12.92 g) were stirred in acetone (120 mL). K₂CO₃ (21 g) wasadded and the mixture was heated in a water bath for 15 minutes. Thereaction mixture was poured into boiling water (800 mL) and the mixturewas left at room temperature for 1 hour. The resultant solid wascollected by filtration and washed with water (3×800 mL) to give a brownpowder (12.8 g). The obtained powder was dissolved in boiling ethylacetate (300 mL), charcoal (5 g) was added and the reaction mixture wasboiled for 10-15 minutes before being filtered through pre-washed (ethylacetate) Celite. The clear, amber solution was concentrated underreduced pressure until crystallisation began. The resultant solid washarvested by filtration, washing with a little ethyl acetate to givecompound 14 (5.5 g, 13.9%) as an off-white powder. m.p. 229-230° C.,[α]_(D) ²⁷ −42° (c 0.5 in CHCl₃). HRMS (ESI) for C₂₄H₂₇O₁₀NNa [M+Na]⁺:m/z calcd 512.1527; measured: 512.1526.

Synthesis 15. DHN-N-acetyl-β-D-glucosaminide (15)

Compound 14 from the previous stage (1.0 g) was suspended in MeOH (3 mL)and deprotected by the method used to make compound 2 (Synthesis 2).This gave compound 15 (330 mg, 44%) as a pale orange solid. m.p.204-205° C., [α]_(D) ²³ −10° (c 1 in MeOH). HRMS (ESI) for C₁₈H₂₂O₇N[M+H]⁺: m/z calcd 364.1391; measured: 364.1388.

Synthesis 16. DHN-dicaprylate (16)

DHN (636 mg) was suspended in a stirred solution of octanoic acid (1.58ml) and DCM (2 ml). Dicyclohexylcarbodiimide (DCCI) (2.2 ml) in DCM (1ml) was added drop wise over 5 minutes. Following addition of the DCCImixture, the DHN dissolved giving a pink coloured solution. After a fewminutes of stirring, a white solid precipitated out of the solution. Thereaction mixture was stirred overnight at room temperature. The whiteprecipitate (urea) was removed by filtration and the filtrate was washedwith 0.5 M NaOH solution (6×10 ml), 1% AcOH solution (1×10 ml) andfinally DI water (2×10 ml). The organic layer was dried (MgSO₄) beforebeing concentrated under reduced pressure to a pale orange oil. The oilwas purified by dissolving in cold hexane (15 ml) with added charcoal(100 mg). After filtration and removal of the solvent under vacuum,compound 16 was obtained as a white waxy oil (970 mg, 59.2%). ¹H NMR:(DMSO-d₆) δ 7.78 (2H, m), 7.63 (2H, s), 7.46 (2H, m), 2.57 (4H, t, J 7.6Hz), 1.76 (4H, q, J 7.4 Hz), 1.46-1.25 (16H, m), 0.89 (6H, m).

Synthesis 17. DHN-phosphate disodium Salt (17)

In a 100 mL round bottom flask MeCN (15.51 g), pyridine (6.96 g) andPOCl₃ (13.49 g) were stirred in an ice/acetone bath. DI water (1.00 g,55.5 mmol) was added dropwise keeping the internal temperature less than20° C. DHN (3.2 g, 19.97 mmol) was added and the mixture was stirred for4 hours at 2° C. The reaction mixture was poured onto ice (50 g) and 10MNaOH (50 mL) was added. The mixture was concentrated under reducedpressure to a pink solid. Following trituration in MeOH, a pink solid(salt) formed and was discarded. The filtrate containing the product andunreacted DHN was concentrated under reduced pressure to dryness. Theobtained solid was triturated in IMS and a pink solid was collected byfiltration. This solid was recrystallized from boiling MeOH (100 mL)containing water (25 mL) using charcoal (1 g). The MeOH was removedunder reduced pressure and replaced with IMS to induce crystallisation.The resultant pink solid was collected by filtration to give the desiredproduct (1.23 g, 21.6%). m.p. >300° C. decomp

Synthesis 18. 1-Nitro-DHN-2′,3′,5′-tri-O-acetyl-β-D-ribofuranoside (18)

In a 1 L 3-neck round bottom flask, a mixture of copper (II) nitrate (60g) and DHN-2′,3′,5′-tri-O-acetyl-β-D-ribofuranoside 1 (20.9 g) wasstirred in DCM (500 mL). After rapid stirring at room temperature for 5min. a very pale yellow colour developed. PTSA (2.0 g) was added and thesolution darkened. The reaction mixture was stirred rapidly at roomtemperature for an additional 50 min. TLC showed a large amount ofremaining starting material. More PTSA (3.0 g) was added and thereaction mixture was stirred rapidly for a further 5 hours. TLC thenshowed no remaining starting material. The reaction mixture was filteredto remove insoluble copper salts and the filtrate was concentrated underreduced pressure to a deep red foaming solid. The solid was purified byflash chromatography using C₆₀ silica gel (1 Kg), eluting withtoluene/acetone 15:1 v/v, collecting fractions of 200 mL. Fractions 3-7were combined and concentrated under reduced pressure to a yellow powder(2.61 g). The yellow powder was dissolved in boiling IMS (50 mL) and theyellow solution left at +4° C. overnight to allow crystallisation.Collected by filtration to give compound 18 as a yellow powder (2.2 g,9.5%). m.p. 151-152° C., [α]_(D) ²³ −88° (c 0.5 in CHCl₃). HRMS (ESI)for C₂₁H₂₅O₁₁N₂ [M+NH₄]⁺: m/z calcd 481.1453; measured: 481.1448.

Synthesis 19. 1-Nitro-DHN-β-D-ribofuranoside (19)

Compound 18 (500 mg) was suspended in MeOH (1.5 mL) and NaOMe solutionin MeOH (2.17 M, 2.5 mL) was added. After overnight reaction thegel-like mixture was concentrated under reduced pressure to a red solid,triturated in IMS (10 mL) and the mixture left at +4° C. overnight. Theresultant gel-like product was collected by filtration and dried undervacuum over P₂O₅ to give compound 19 (137.5 mg, 38%) as a yellow solid.m.p. >170° C. decomp. [α]_(D) ²² −73° (c 0.23 in water). HRMS (ESI) forC₁₅H₁₉O₈N₂ [M+NH₄]⁺: m/z calcd 355.1136; measured: 355.1140.

Synthesis 20 (Reference). 1,4,6,7-Tetrabromo-DHN (20)

This was made from DHN (18 g) and bromine (23.2 mL) by the method ofZincke and Fries [loc. cit.]. This produced compound 20 (36.19 g, 67%)as a very pale pink solid. m.p. 240-242° C.

Synthesis 21 (Reference). 6,7-Dibromo-DHN (21)

The title compound was made by treating 1,4,6,7-tetrabromo-DHN 20 (8 g)with tin (II) chloride (32 g) according to the conditions described byZincke and Fries [loc. cit.]. This produced 21 (3.65 g, 68%) as a whitepowder, m.p. 211-213° C.

Synthesis 22 (Reference).6,7-Dibromo-DHN-2′,3′,5′-tri-O-acetyl-β-D-ribofuranoside (22)

In a 100 mL round bottom flask, 6,7-dibromo-DHN 21 (3 g),1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (3.3 g) and 3 Å mol. sieves (1g) were stirred in DCM (30 mL) for 10 min. BF₃.etherate (3 mL) was thenadded. After 15 min. a thick white precipitate had formed. The reactionmixture was poured into a mixture of DCM (200 mL) and sat. NaHCO₃ (200mL). The white solid dissolved. The organic layer was separated andwashed with sat. NaHCO₃ (2×200 mL) and DI water (200 mL), dried (MgSO₄)and concentrated under reduced pressure to give a white solid. The solidwas triturated in IMS (50 mL) and the resultant solid collected byfiltration to give compound 22 (4.42 g, 81%). m.p. 185-186° C., [α]_(D)²³ −51° (c 1 in CHCl₃). HRMS (ESI) for C₂₁H₂₄O₉NBr₂ [M+NH₄]⁺: m/z calcd591.9812; measured: 591.9805.

Synthesis 23. 6,7-Dibromo-DHN-β-D-ribofuranoside (23)

Compound 22 (1.0 g) was suspended in MeOH (3 mL) and deprotected by themethod used to make compound 2 (Synthesis 2) to give compound 23 (697mg, 89%). m.p. decomp. ≥240° C., [α]_(D) ²³ −90° (c 0.5 in MeOH). HRMS(ESI) for C₁₅H₁₈O₆NBr₂ [M+H]⁺: m/z calcd 465.9495; measured: 465.9495.

Synthesis 24 (Reference).6,7-Dibromo-DHN-2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranoside (24)

6,7-Dibromo-DHN 21 (6 g) and acetobromogalactose (10.23 g) were stirredin acetone (1 mL). NaOH (900 mg) in DI water (20 mL) was added in oneportion. The reaction mixture was stirred overnight at room temperatureat which point TLC showed no remaining acetobromogalactose. The reactionmixture was poured into DCM (500 mL) and sat. NaHCO₃ (500 mL). The DCMlayer was separated and washed with sat. NaHCO₃ (6×500 mL) and DI water(500 mL), dried (MgSO₄) and concentrated under reduced pressure to givea brown oil (12.37 g). The obtained oil was purified by flashchromatography using C₆₀ silica gel (550 g) eluting with toluene/acetone10:1 v/v and collecting fractions of 200 mL. Fractions 5-17 werecombined and concentrated under reduced pressure to a green foaming oil(6.6 g). TLC showed that this oil contained the desired product compound24 and unreacted 6,7-dibromo-DHN 21. The oil was dissolved in DCM (150mL) and washed with 1M NaOH (150 mL) and DI water (150 mL) before beingdried (MgSO₄) and concentrated under reduced pressure to give compound24 (4.6 g, 37%) as a pale green foaming solid, [α]_(D) ¹⁹ +20° (c 0.5 inacetone). HRMS (ESI) for C₂₄H₂₈O₁₁NBr₂ [M+NH₄]⁺: m/z calcd 664.0024;measured: 664.0023.

Synthesis 25. 6,7-Dibromo-DHN-β-D-galactopyranoside (25)

Compound 24 (4.35 g) was deprotected in a similar manner to that used tomake compound 2 (Synthesis 2) except that the solution was neutralisedwith Amberlite® IR120H⁺ resin. The powder was purified byrecrystallization from MeOH/water 1:1 v/v (300 mL) to give compound 25(1.89 g, 59%) as a white solid. m.p. 256-257° C., [α]_(D) ²⁵ −64° (c0.25 in acetone/water 1:1 v/v). HRMS (ESI) for C₁₆H₁₆O₇Br₂ [M+H]⁺: m/zcalcd 476.9190; measured: 476.9203.

Synthesis 26 (Reference).6,7-Dibromo-DHN-2′,3′,4′,6′-tetra-O-acetyl-β-D-glucopyranoside (26)

This was made from 6,7-Dibromo-DHN 21 (6 g) and acetobromoglucose (10.23g) by essentially the same method used for the analogousgalactopyranoside 24 (Synthesis 24). The title compound 26 was obtainedas a white solid (3.16 g, 25%). m.p. 166-167° C., [α]_(D) ²⁵ −28° (c 0.5in acetone). HRMS (ESI) for C₂₄H₂₈O₁₁NBr₂ [M+NH₄]⁺: m/z calcd 664.0024;measured: 664.0025.

Synthesis 27. 6,7-Dibromo-DHN-β-D-glucopyranoside (27)

Compound 26 (3 g) was suspended in MeOH (8 mL) and deprotected by theaddition of NaOMe solution in MeOH (2.17 M, 3 mL). Work-up gave compound27 (2.04 g, 92%) as a very pale green solid. m.p. decomp. >290° C.,[α]_(D) ²⁵ −168° (c 0.25 in water). HRMS (ESI) for C₁₆H₁₅O₇Br₂ [M+H]⁺:m/z calcd 476.9190; measured: 476.9186.

Synthesis 28 (Reference). 6,7-Dibromo-DHN-2′,3′,4′-tri-O-acetylβ-D-glucuronide-6′-methyl ester (28)

α-D-2,3,4-Tri-O-acetyl-β-D-glucuronyl-trichloroacetimidate-6-methylester (10 g) and 6,7-dibromo-DHN 21 (6 g) were stirred in DCM (100 mL).BF₃.etherate (˜200 μL) was added. The reaction mixture was stirred atroom temperature for 30 min. before being poured into a mixture of DCM(500 mL) and sat. NaHCO₃ (500 mL). The DCM layer was separated andwashed with sat. NaHCO₃ (4×500 mL) and DI water (500 mL), dried (MgSO₄)and concentrated under reduced pressure to give a pink solid. Theobtained solid was triturated in IMS (100 mL) and left at +4° C.overnight. The resultant solid was harvested by filtration to givecompound 28 (2.89 g, 24%) as a white solid. m.p. 176-177° C., [α]_(D) ²⁵−16° (c 0.25 in acetone). HRMS (ESI) for C₂₃H₂₆O₁₁NBr₂ [M+NH₄]⁺: m/zcalcd 649.9867; measured: 649.9868.

Synthesis 29. 6,7-Dibromo-DHN-β-glucuronide CHA Salt (29)

Compound 28 (2.5 g) was deprotected with NaOH (870 mg) in a mixture ofDI water (11.5 mL) and acetone (23 mL). Work-up as for compound 12a withpro-rata quantities gave compound 29 as a white solid (2.16 g, 95%).m.p. 221-224° C., [α]_(D) ²⁵ −159° (c 0.25 in water). HRMS (ESI) forC₁₆H₁₃O₈Br₂ [M+H]⁺: m/z calcd 490.8983; measured: 490.8976.

Synthesis 30 (Reference). DHN-6-sulfonicacid-2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranoside sodium Salt (30)

DHN-6-sulfonic acid sodium salt (5 g) was dissolved in a solution ofNaOH (850 mg) in DI water (150 mL). Acetobromogalactose (7.06 g),tetrabutylammonium bromide (6.75 g) and DCM (8.7 g) were added and thetwo-phase reaction mixture was stirred overnight at ambient temperature.TLC analysis showed no remaining acetobromogalactose. The reactionmixture was diluted with additional DCM (100 mL) and the organic layerwas separated before being washed with DI water (300 mL), dried (MgSO₄),and concentrated under reduced pressure to an oil (15.83 g). Theisolated oil was purified by flash chromatography using C₆₀ silica (600g), eluting with DCM/MeOH. Fractions 7-20 were combined and concentratedunder reduced pressure to an oil which was triturated in ethyl acetate(30 mL). The resultant white solid was collected by filtration to giveprotected mono-galactoside 30 (4.77 g, 49.68%). m.p. 131-133° C.,[α]_(D) ²³ +5° (c 0.592 in acetone). ¹H NMR (DMSO-d₆): δ 9.73 (1H, s),7.90 (1H, s), 7.55 (1H, dd), 7.45 (1H, m), 7.40 (1H, s), 7.16 (1H, s),5.47 (1H, d), 5.32 (1H, broad s), 5.25 (2H, m), 4.43 (1H, m), 4.10 (1H,m) 2.13, 1.99, 1.96, 1.91 (4×3H, 4×s). ¹H NMR spectroscopy confirmed theobtained solid was the mono-galactoside, although which of the twopossible isomers could not be determined from the NMR data.

Synthesis 31. DHN-6-sulfonic acid-β-D-galactopyranoside sodium Salt (31)

Compound 30 (1 g) was suspended in MeOH (3 mL), 2.17 M NaOMe (0.5 mL)was added. Additional 2.17 M sodium methoxide (3 mL, 3.3 mmol) was addedto aid crystallisation of the sodium salt. After complete deprotection(by TLC) the mixture was concentrated under reduced pressure andtriturated in IMS (10 mL). The solid was collected by filtration andwashed with a little IMS to afford compound 31 (462 mg, 62%).m.p. >2300° C. decomp, [α]_(D) ²³ −73° (c 0.26 in water). ¹H NMR(DMSO-d₆): δ 7.45 (1H, s), 7.27 (1H, d), 7.09 (1H, s), 6.98 (1H, dd),6.41 (1H, s), 4.55 (1H, d), 3.63 (1H, d), 3.52 (3H, m), 3.31 (2H, m). ¹HNMR spectroscopy confirmed the obtained solid was the mono-galactoside,although which of the two possible isomers could not be determined fromthe NMR data.

Synthesis 32. DHN-6-sulfonic acid-phosphate trisodium Salt (32)

In a 100 mL round bottom flask MeCN (15.51 g), pyridine (6.96 g) andPOCl₃ (13.49 g) were stirred in an ice/acetone bath. DI water (1.00 g,55.5 mmol) was added dropwise keeping the internal temperature less than20° C. DHN-6-sulfonic acid sodium salt (5.24 g) was added and themixture was stirred for 4 hours at 2° C. The reaction mixture was pouredonto ice (100 g) and 10M NaOH (35 mL) was added. The mixture wasconcentrated under reduced pressure to give an orange solid which wastriturated in MeOH (200 mL). The resultant solid (33.4 g) was mainlysalt and was discarded. The filtrate was concentrated under reducedpressure to an orange solid (4.44 g). This was dissolved in hot DI water(20 mL), filtered, and IMS (200 mL) was added to the clear brownfiltrate. A brown oil separated out of the mixture. After decanting theIMS/water solution from the oil, a pale cream precipitate formed in thissolution. It was removed by filtration, and this filtrate was nowrecombined with the previously isolated brown oil. Concentration of thismixture under reduced pressure gave a brown foam. This foam was heatedwith MeOH (75 mL) in an ultrasonic bath; a fine hygroscopic brown solidwas removed by filtration and the clear brown filtrate was concentratedunder reduced pressure to a solid. Trituration of the solid in IMS (30mL) gave a mixture of isomers of title compounds (32) as an off-whitesolid (1.17 g, 14.7%). m.p. >400° C.

The invention claimed is:
 1. A composition comprising a) an enzymesubstrate of formula I:

wherein Y is an enzyme cleavable group; Z is H, a metal cation ornon-metal cation, acyl or the same enzyme cleavable group as Y; R² andR³ are each independently selected from the group consisting of H, OH,C₁-C₈ alkyl, C₂-C₂₄ acyl, halogen and nitro, provided that if Z is H ora salt, then R² must not be OH; R¹ is C₁-C₈ alkyl, halogen, OH, NO₂,C₂-C₂₄ acyl, or —SO₃X, where X is H, a metal cation or a non-metalcation; and n is 0-4; b) a metal compound such that a product ofenzymatic substrate cleavage is capable of chelating a metal ion of themetal compound, thereby forming a colored compound, wherein the metalcompound is an iron salt; and c) microbial growth nutrients.
 2. Thecomposition according to claim 1, wherein the microbial growth nutrientsinclude a carbon source, a nitrogen source, amino acids, salts, vitaminsand/or cofactors.
 3. The composition according to claim 1, wherein themolar ratio of enzyme substrate to metal compound is in the range 0.05to
 50. 4. A composition comprising a) an enzyme substrate of formula I:

wherein Y is an enzyme cleavable group; Z is H, a metal cation ornon-metal cation, acyl or the same enzyme cleavable group as Y; R² andR³ are each independently selected from the group consisting of H, OH,C₁-C₈ alkyl, C₂-C₂₄ acyl, halogen and nitro, provided that if Z is H ora salt, then R² must not be OH; R¹ is C₁-C₈ alkyl, halogen, OH, NO₂,C₂-C₂₄ acyl, or SO₃X, where X is H, a metal cation or a non-metalcation; and n is 0-4; and b) a metal compound that is an iron salt. 5.The composition according to claim 4, further comprising microbialgrowth nutrients.
 6. The composition according to claim 5, wherein themicrobial growth nutrients include a carbon source, a nitrogen source,amino acids, salts, vitamins and/or cofactors.
 7. The compositionaccording to claim 4, wherein the molar ratio of enzyme substrate tometal compound is in the range 0.05 to
 50. 8. A composition comprisingan enzyme substrate of formula II

wherein one of the following applies: i) m=0, R⁴═R⁵═Z¹═H, Y¹ is selectedfrom the group consisting of D-glucuronyl and D-ribofuranosyl; ii) m=2,each R⁶ is Br, R⁴═R⁵═H, Z¹═H; Y¹ is glycosyl or phosphate; iii) m=1, R⁶is —SO₃X, X is H or M⁺ wherein M⁺ is an alkali metal cation or anon-metal cation, Y¹ is glycosyl or phosphate and R⁴═R⁵═Z¹═H; iv) m=0,R⁴═NO₂, R⁵═Z¹═H, Y¹=glycosyl.
 9. The composition of claim 8, furthercomprising a metal compound, a metal ion of which is chelatable by theproduct of enzyme cleavage of the group Y¹ and, in the case of optioniv), also of the group Z¹, from the substrate.
 10. The compositionaccording to claim 9, wherein the metal compound is an iron salt. 11.The composition according to claim 9, further comprising microbialgrowth nutrients.
 12. The composition according to claim 11, wherein themicrobial growth nutrients include a carbon source, a nitrogen source,amino acids, salts, vitamins and/or cofactors.
 13. The compositionaccording to claim 9, wherein the molar ratio of enzyme substrate tometal compound is in the range 0.05 to 50.